Agricultural yields have historically been threatened and often destroyed by extremes of temperature as well as by drought and flood. With the changes in environment attributed to global warming, extreme weather conditions are expected to increase. Agricultural yields are also threatened in many important agricultural areas due in part to long-term effects of farming practices on the land. For example, in many areas, the chemical composition of the soil has actually been altered by the accumulation of salts such that increased salinity is now a hindrance to optimal plant growth. See Boyer (1982) Science 218: 443–448. Other stresses of economic importance to agriculture include those imposed by pathogens as well as nitrogen starvation, ozone, and increased concentrations of heavy metals in the soil.
Stress responses in plants and ways of improving stress responses have been widely studied. Many links have been discovered between stress responses generally and pathogen attack, so approaches to improving stress responses often have a positive impact on plant resistance to pathogens. See Farmer (2000) Genome Biol. 1(2): reviews 1012.1–1012.3. For example, in Arabidopsis, the response to ozone involves the same salicylic acid-dependent pathway that is involved in microbial pathogen resistance. See Sharma and Davis (1997) Free Radic. Biol. Med. 23(3): 480–88.
Salt tolerance in plants has also been widely studied. One approach to improving salt tolerance is alteration of the metabolic responses of crop plants. Most genes that show increases in activity or amount in response to high salt concentration also are induced by abscisic acid (“ABA”), and it has also been shown that many stress-inducible genes in plants, for example, oxalate oxidase, are also induced in the normal course of development.
Because plants are subject to a number of environmental stresses, regulation of expression of helpful gene products in response to stress may decrease damage resulting from such environmental stresses and improve crop performance and yields. Conventional plant breeding has improved yields for many crops grown under stressful conditions. However, continued efforts to improve plant response to stress has led many to the conclusion that further improvements will come not from conventional plant breeding but rather from the more precisely focused changes made possible by genetic engineering. See Smirnoff and Bryant (1999) Nature Biotechnol. 17: 229; Ceccarelli and Grando (1996) Plant Growth Reg. 20: 149–155.
Experiments in improving plant stress tolerance have shown some success. For example, constitutive overexpression of DREB1A, a stress-regulatable transcription factor from Arabidopsis, conferred an improved response to stresses including dehydration, freezing, and increased salinity. Kasuga et al. (1999) Nature Biotechnol. 17, 287–291. However, there was a significant drawback of the constitutive expression of the transgene: under normal growth conditions (i.e., without stress), plant growth was severely impaired. Fortunately, when the transgene was expressed only in response to stresses such as cold and water stress, the plants exhibited stress tolerance without the growth impairment resulting from constitutive expression. Thus, appropriate expression of helpful transgenes has potential for the improvement of stress tolerance of crops. Accordingly, there is a need for transgenes which can improve plant response to stress as well as a need for mechanisms to appropriately express these transgenes.
On the other hand, expression of certain genes known to be related to stress tolerance may improve plant growth and yield even in the absence of stress. For example, altering the ABA and sugar signaling of a plant may improve yield under non-stress conditions. Thus, there is a need for controlled and targeted manipulation of stress-related gene expression for improved performance of plants in the absence of stress.
Stress-inducible genes of plants play an important role in other aspects of plant growth, development, and defense mechanisms, as suggested by their complex patterns of regulation and roles in response to stress. See, e.g., Berna and Bernier (1999) Plant Mol. Biol. 39(3): 539–549; Roitsch (1999) Curr. Opin. Plant Biol. 2(3): 198–206. For example, wheat oxalate oxidase causes release of hydrogen peroxide in the apoplast in response to stress as well as during normal development. Other stress response genes also play a role in non-stress physiological processes, such as berry ripening. Davies and Robinson (2000) Plant Physiol. 122(3): 803–812. The fact that stress-response genes play roles in many other physiological processes highlights the importance of a narrowly tailored genetic alteration in improving stress response.
Stress responses in plants often include dramatically increased expression of sets of genes. However, stress responses in plants are not exclusively a matter of increased gene expression, but can be more complex. For example, studies of potato show that wounding of the potato tissue led to increased gene expression which resulted in tissue repair. In contrast, in potato subjected to impact injury and loss of membrane integrity, the enzyme polyphenol oxidase is redistributed at the subcellular level. See Partington et al. (1999) Planta 207(3): 449–460.
Abscisic acid (“ABA”) is a plant hormone important in modulating the response of plants to stresses such as drought, salt, and cold stress. ABA is also important in many aspects of plant metabolism and development, including seed development, response of tissues to sugar signals (Rook et al. (2001) Plant J. 26: 421–433), seed dormancy, seed desiccation tolerance, storage of seed proteins and lipids, and stomatal movement. See Merlot and Giraudat (1997) Plant Physiol. 114: 751–757. Studies of the relationship between ABA and plant responses to sugar have revealed similar responses of plants to both signals, and that both ABA and sugar play a role in germination, photosynthesis, and development. See Huijser et al. (2000) Plant J. 23: 577–585; Arenas-Huertero et al. (2000) Genes Dev. 14: 2085–2096; Laby et al. (2000) Plant J. 23: 587–596.
A handful of genes important to the response of plants to ABA have been cloned, including genes which encode transcriptional regulators from maize (the Viviparous1 gene, or Vp1). The Arabidopsis ABI4 gene also appears to be a transcriptional regulator and is thought to play a general role in ABA response. See Finkelstein et al. (1998) Plant Cell 10: 1043–1054 (1998); Arenas-Huertero et al (2000) Genes Dev. 14: 2085–2096. Other ABA-response genes have been identified as protein phosphatase 2C genes and a farnesyl transferase gene. See Finkelstein et al. (1998) Plant Cell 10: 1043–1054.
Mutations in ABI4 have been isolated in screens for mutations showing: increased salt tolerance (Quesada et al. (2000) Genetics 154: 421–436); seed germination in the presence of high levels of ABA; seed germination in the presence of high salinity (Quesada et al. (2000) Genetics 154: 421–436); and seed germination in the presence of high levels of glucose (Huijser et al. (2000) Plant J. 23: 577–585, and references cited therein). Thus, ABA-insensitive mutants have defects in dormancy of seed. In addition, ABI4 mutant plants have altered seed protein composition. These defects are consistent with the proposed roles for ABI4 in seed development such as regulating seed responses to ABA. See Finkelstein et al. (1998) Plant Cell 10: 1043–1054; Soderman et al. (2000) Plant Phys. 124: 1752–1765. However, expression analysis of Arabidopsis has showed that AtABI4 expression is not limited to seed.
Arabidopsis plants which contain mutations in the ABI4 gene are typically insensitive to ABA and show defects in stress responses, including those involving root growth, stomatal regulation, and regulation of stress-induced genes. ABI4 mutants have been isolated in screens for plant mutants causing a defect in feedback inhibition of photosynthesis and induction of storage processes which normally result in the presence of high levels of sugar. See Rook et al. (2001) Plant J. 26: 421–433. ABI4 mutants in Arabidopsis have also been identified as insensitive to certain sugars, indicating that ABI4 plays a role in a sugar-response pathway that contributes to regulation of photosynthesis. See Huijser et al. (2000) Plant J. 23: 577–585.
The Arabidopsis ABI4 gene has been cloned and sequenced. See Finkelstein et al. (1998) Plant Cell 10: 1043–1054. The predicted protein product of the ABI4 gene shows homology to plant transcriptional regulators which contain the conserved APETALA2 DNA-binding domain. However, outside this conserved region, the Arabidopsis ABI4 gene shared a much lower degree of identity with these other APETALA2 genes (around 40%; see Finkelstein et al. (1998) Plant Cell 10: 1043–1054). The null alleles of ABI4 isolated in screens for salt-tolerant mutants of Arabidopsis were shown to be missing the APETALA2 DNA binding domain.
Other motifs important to stress responses include a motif called DREB/CBF. See Smirnoff and Bryant (1999) Nature Biotechnol. 17: 229. Some genes important to stress response to freezing and water stress have been shown to contain the DREB/CBF motif. See Close (1997) Physiol Planta 100: 291–296. Similarly, studies have led to the discovery of cis-acting regulatory sequences important in the stress response, such as ABA response elements (Shen and Ho (1995) Plant Cell 7(3): 295–307) and the C-repeat/dehydration responsive element (DRE). See Stockinger et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1035–1040. In Arabidopsis, Stockinger et al. isolated a CBF1 protein which binds to the DRE and thereby stimulates transcription in response to water stress and low temperatures. CBF1 also contains an APETALA2 domain. Stockinger et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1035–1040.
Nevertheless, water stress around the time of flowering has a significant negative impact on maize yield. One potential strategy to reduce these effects is to engineer the maize plant to improve the stress response. Previously characterized regulatory sequences are present in only a subset of response genes. Thus, there is a need in the area for the tools to create such improvements, including suitable combinations of promoters, response elements, and transcription factors to appropriately control expression of helpful genes.